TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis

Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

Introduction to investigate the roles of CsEVs in regulating the activation of TLR3 and the contribution to biliary injuries.

Ethics statement
All experimental procedures involving animals were conducted in compliance with Chinese legal standards, and the experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University. (IACUC permit number: 20160612).

Animals
TLR3 -/and WT female C57BL/6 mice (aged 6-8 weeks) were housed under constant temperature and pathogen-free animal conditions for 12 h on a dark/light cycle with sterile water and normal mouse chow.

Collection of C. sinensis and CsEVs
The C. sinensis excretory/secretory proteins (CsESPs) were immediately used to isolate CsEVs [9]. The C. sinensis adults were isolated from bile duct of metacercariae infected WT C57BL/6 mice: The mice were euthanized after 35 days post infection (dpi) and disinfected with 75% alcohol for 15 min. Then, the mice were transferred to a sterile platform, and aseptic procedures were performed throughout the trial. Carefully isolate the liver, soak it in sterile PBS, and open the bile duct. Gently pressing the liver using ophthalmic forceps and picking out the adults.
The isolate adults were incubated at density of five parasites per mL of serum-free RPMI-1640 at 37˚C for 6 h. The incubated supernatant was collected and centrifuged at 280×g and 2 000×g, continuously. Then the supernatant was filtered through a syringe filter (0.22-μm, Millipore, Massachusetts, USA). Then a slightly modified differential centrifugation method was used to collect CsEVs from the CsESPs [12]. Briefly, CsESPs were centrifuged at 10 000 ×g for 1 h, the supernatant was collected then passed through a filter (0.22 μm). The filtered liquid was centrifuged at 100 000 ×g for 1 h, and the sediment was collected. The precipitate was dissolved in sterile phosphate-buffered saline (PBS) and the CsEVs concentration was determined using BCA. Then, CsEVs on copper grids were negatively stained using carbon and 3% phosphotungsticacid and observed under a transmission electron microscope (TEM) (HITACHI, Tokyo, Japan). And a fraction of CsEVs were treated with RNase A (Solarbio, Beijing, China) to remove dsRNAs in CsEVs-free ESPs (rCsEVs).

Isolation of mouse BECs
The biliary tree was obtained by removing liver membranes and hepatocytes through perfusion using HEPS solution (Biosharp, Anhui, China) (with 0.05mg/mL collagenase IV(Sigma-Aldrich, Missouri, US)) as conventional methods [13,14]. Then, the isolated biliary tree was divided into small pieces and further digested in 0.05mg/mL collagenase IV solution for 30 min under shaking conditions, and were centrifuged at 800 ×g for 10 min. The precipitate was retained, digested by 0.25% trypsin-digested (VivaCell, Shanghai, China) at 37˚C for 10min. After centrifugation (800 ×g for 10 min), trypsin was discarded and RPMI-1640 medium with 5% serum was added to stop digestion. After repeated washing and centrifugation, the BECs were resuspended using RPMI-1640 medium (with 5% serum) and filtered through a 75μm aperture metal sieve. Cell number and viability were assessed using a 0.4% trypan blue solution (Procell, Wuhan, China) and BECs with 3 ×10 5 were plated incubated at 37˚C with 5% CO 2 in 6 well tissue culture plates. To investigate CsEVs functions, The BECs were plated and treated with the CsEVs (50 μg/mL), rCsEVs (50 μg/mL) or PBS cultures for 2 h or 18 h at 37˚C with 5% CO 2 , respectively.

C. sinensis-caused liver fibrosis mouse model
TLR3 -/and WT mice were inoculated with 200 C. sinensis metacercariae by gavage in 200 μL of PBS (pH 7.4), with mice administered 200 μL of PBS as the control. Poly (I:C)-intervened WT mice were injected intraperitoneally twice with poly (I:C) (10 mg/kg) (InvivoGen, California, US) at 0, 10, and 20 dpi. Weight and mortality rates were monitored daily until sacrifice. Mice were euthanized at 7, 15, and 35 dpi to examine the liver lesions and intrahepatic parasite burdens. Mouse faeces were collected daily after infection. Rice grain sized faeces were placed on a slide, added 50 μL of saline then covered with a coverslip to examine the eggs under the microscope. The liver tissues were isolated and used for cytokine detection, quantitative realtime PCR (RT-qPCR), western blotting, histological observation, Masson staining, and immunohistochemical staining.

RT-qPCR analysis
Total RNAs from WT mouse livers and BECs was extracted using the TRIzol reagent and the cDNA was synthesized from the total RNA using reverse transcriptase prior to the PCR step. Then the mRNA levels were determined by RT-qPCR followed the conditions and procedures of Green qPCR Mix (Monad, Suzhou, China). The primer sequences used were TLR3 F: 5-AAGACAGAGACTGGGTCTGGG-3, R:5-AAGGACGCCTGCTTCAAAGT-3; and GAPDH F: 5-CCATGTTTGTGATGGGTGTG-3, R: 5-CCTTCTTG ATGTCATCATAC-3 [16]. The data were normalized to GAPDH and the mRNA level of all experimental groups was presented in terms of numerical vales calculated using (delta)(delta)C/t equation.

Cytokine detection
Liver tissues of TLR3 -/and WT mice were lysed into cell suspensions, and the supernatant was stored for cytokine detection. WT and TLR3 -/mouse BECs were incubated with CsEVs (50 μg/mL) for 18 h, and poly (I:C) (30 μg/mL) was used as the positive control [17]. To investigate the role of p38 and ERK signaling pathways in regulating cytokine production, the WT BECs were pretreated with p38 or ERK inhibitors (Sigma-Aldrich, Missouri, US) for 60 min at 37˚C, with untreated cells as the control. Then the cells were co-stimulated with CsEVs for 18 h. The secretion levels of cytokines in the liver tissues and BECs supernatant were detected using ELISA kits (IL-4/IL-6/IFN-γ/TGF-β1/TNF, Thermo Scientific, Massachusetts, US) [17].

Histology observation
Liver tissues from the same positions were removed and embedded in paraffin. The tissue sections were sliced to a thickness of 3 μm, deparaffinized with xylene, and stained with hematoxylin and eosin. Hepatic injury and inflammation were thoroughly documented under a microscope and evaluated using the hepatic histological activity index (HAI) [18,19].

Masson staining
The paraffin-embedded liver tissues were subjected to the same procedure until routine deparaffinization, and the tissue sections were stained with Masson's Trichrome Stain Kit (Solarbio, Beijing, China). The positive area of collagen fibers was scanned and quantified using the Image-Pro Plus software (Media Cybernetics, Massachusetts, USA).

Immunohistochemistry
Liver tissue was sliced into 5 μm sections and analyzed using routine immunohistochemistry. After peroxidase removal and antigen repair, the liver tissue sections were treated with FBS at room temperature for 30 min and then incubated overnight with CK-19 and α-SMA antibodies at 4˚C, followed by incubation with HRP-conjugated antibodies. After counterstaining with hematoxylin, the tissue sections were mounted with neutral gum. The positive area of immunohistochemistry was digitized and analyzed using the Image-Pro Plus software. Antibody information is presented in S1 Table. Immunofluorescence Adult C. sinensis was isolated, transferred to a cell culture dish, and incubated in 1640 medium for 12 h. After washing with sterile PBS, the worms were fixed and permeabilized, and dsRNAs was labeled with J2 antibody and FITC-labeled fluorescent secondary antibody. The nuclei were stained with Hoechst (Sigma-Aldrich, Missouri, US), and the subcellular localization of dsRNA was observed using the Live Cell Imaging System (Olympus, Tokyo, Japan). Antibody information is presented in S1 Table.

Western blot
Liver tissues and BECs were collected and resuspended in RIPA lysis buffer containing PMSF (1:100) (Boster Bio, California, USA). The SDS-PAGE and membrane transfer tests were executed as previously reported [17]. The membranes were incubated overnight at 4˚C with primary antibodies (Phosphorylated protein p65, ERK, Smad2/3, p38 and Total protein p65, ERK, Smad2/3, p38). Membranes were incubated with secondary antibody for 1 h at room temperature after three washes with PBST. An ECL-Chemiluminescence meter was used to visualize the protein (Clinx Science Instruments Co., Ltd., Shanghai, China). The protein expression level was quantified using ImageJ (National Institutes of Health, Bethesda, Maryland, USA) and the relative gray values of phospho-p65/GAPDH, phospho-Smad2/3/GAPDH, phospho-ERK/GAPDH and phospho-p38/GAPDH were calculated using Excel (Microsoft Corp., Redmond, WA, USA) software, respectively [9]. Antibody information is presented in S1 Table. Dot Immunobinding Assay (DIBA) The dot immunoassay was performed according to the previous protocol [20]. Briefly, the sheared nitrocellulose membranes (NCM) were placed into reaction wells and 50 μL of CsEVs (1 mg or 0.1mg/mL), Poly (1:C) (30 μg/mL), and PBS were dropped onto NCM, and acted at room temperature for 30 min, and the remaining liquid was discarded. Then NCM was sealed with 5% milk, and then incubated overnight with J2 antibody at 4˚C. The membrane was incubated at room temperature with HRP-link antibodies for 30 min after three washes with PBS, and the results were viewed using an imaging system. Antibody information is presented in S1 Table.

Statistical analysis
GraphPad Prism Software (version 6.01) was used to conduct Tukey tests (T-tests) and twoway ANOVA on the data set and generated pictures (GraphPad Software Inc, California, US). The experiment data was obtained from three independent experiments, with the results expressed as the mean ± SEM. Significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

TLR3 deficiency caused more severe clonorchiasis in C. sinensis-infected mice
RT-qPCR revealed a significant elevation of TLR3 mRNA in the liver of C. sinensis-infected WT mice at 7 dpi, 15dpi, and 35 dpi ( Fig 1A). C. sinensis-infected TLR3 -/mice showed lower weight and a reduced survival rate compared to C. sinensis-infected WT mice (Fig 1B and 1C). The survival rate of C. sinensis-infected TLR3 -/mice was reduced by approximately 10% compared with that infected WT mice ( Fig 1C). There was a significant difference in the number of intrahepatic parasites between C. sinensis-infected WT and TLR3 -/mice, and the number of C. sinensis adults in TLR3 -/mice was significantly higher than that in WT mice ( Fig 1D).

TLR3 deficiency aggravated C. sinensis-induced bile duct lesions and liver inflammation
Significant pathological changes, including hepatomegaly, cholestasis, jaundice, dilated protrusions of bile ducts, and connective tissue hyperplasia, were observed in the livers of C. sinensis- infected WT mice. TLR3 deficiency deteriorated the liver lesions induced by C. sinensis, with more pronounced bile duct degeneration, connective hyperplasia, and cholestasis than in WT mice (Fig 2A).
We observed pathological injury in the livers and bile ducts of C. sinensis-infected TLR3 -/and WT mice at 7,15, and 35 dpi. There were hepatocellular necrosis foci, large numbers of inflammatory cells gathered around the necrotic foci and portal veins, cholangiectasis, and BECs proliferating and disorganized in the livers of C. sinensis-infected WT mice. Liver inflammation and biliary injury in C. sinensis-infected mice increased with infection duration (Fig 2B-2D). The livers of C. sinensis-infected TLR3 -/mice showed more severe hepatic necrosis foci and bile duct degeneration, as well as more inflammatory cells compared to WT mice (Fig 2B-2D).
The expression levels of IL-6, IL-4, TNF and IFN-γ in livers of C. sinensis-infected WT mice were significantly increased compared to PBS treatment mice at 7,15, and 35 dpi (Fig 2E-2H). IL-6, TNF, and IL-4 were significantly increased in the livers of TLR3 -/mice compared to WT mice (Fig 2E-2G). In contrast, IFN-γ expression was remarkably reduced in the livers of TLR3 -/mice compared to that in C. sinensis-infected WT mice (Fig 2H).

TLR3 deficiency aggravated liver fibrosis caused by C. sinensis
Liver fibrosis in C. sinensis-infected WT and TLR3 -/mice was observed using Masson staining. The results showed that C. sinensis caused severe liver fibrosis, with persistent accumulation of collagen fibers around bile ducts at 7 dpi and 15 dpi, and even developed liver fibrosis at 35 dpi in WT mice (Fig 3A and 3B). The deposition of collagen fibrils in the liver of TLR3 -/mice was more severe and more rapid at 7 dpi, 15 dpi, and 35 dpi than in WT mice (Fig 3A and 3B). These results suggest that TLR3 attenuates the liver fibrosis caused by C. sinensis.

TLR3 deficiency promoted the activation of TGF-β/Smad pathway and myofibroblasts induced by C. sinensis
We examined myofibroblast activation in the livers of C. sinensis-infected mice via α-SMA immunohistochemistry. The results showed that α-SMA-positive myofibroblasts appeared around the bile duct at 7 dpi and progressively increased at 15 dpi and 35 dpi in the livers of C. sinensis-infected WT mice (Fig 4A and 4B). The number of positive myofibroblasts was significantly higher in the livers of C. sinensis-infected TLR3 -/mice than that in C. sinensis-infected WT mice at 15 dpi and 35 dpi (Fig 4A and 4B).
The TGF-β/Smad pathway was detected in the livers of C. sinensis-infected mice at 7, 15, and 35 dpi. The results showed that C. sinensis infection significantly promoted TGF-β1 expression at 7, 15, and 35 dpi ( Fig 4C) and increased the Smad2/3 phosphorylation at 15 and 35 dpi in the livers (Fig 4D and 4E). TLR3 deficiency further increased the expression of TGF-β1( Fig 4C) and phosphorylation level of Smad2/3 at 15 and 35 dpi (Fig 4D and 4E). These results suggest that host TLR3 contributes to reducing C. sinensis-induced the activation of TGF-β/Smad pathway and myofibroblast, thereby alleviating liver fibrosis.

CsEVs regulated the production of proinflammatory cytokines via TLR3-mediated p38 and ERK pathways
To explore the TLR3-mediated mechanism of C. sinensis-induced liver fibrosis, we isolated CsEVs and examined the phosphorylation of inflammatory pathways and cytokine expression in mouse BECs stimulated with CsEVs. The results showed that CsEVs were slightly concave The data were obtained from 30 mice in each group, and three independent repeated trials were conducted. *p<0.05, ***p<0.001, two-way ANOVA were performed to compare the samples.
https://doi.org/10.1371/journal.pntd.0011325.g002 PLOS NEGLECTED TROPICAL DISEASES TLR3 alleviates C. sinensis-induced liver fibrosis and spherical in shape, with a size of approximately 80-120 nm (Fig 5A and 5B). CsEVs were rich in dsRNA and significantly activated the transcriptional level of TLR3 in BECs (Fig 5C  and 5D). Further, rCsEVs co-incubated with BECs, RT-PCR detected the expression of TLR3 mRNA. The results showed that rCsEVs could significantly activate the expression of TLR3 in BECs, and there was no significant difference from untreated CsEVs co-incubation BECs (S1 Fig). We performed immunofluorescence staining of adults using the J2 antibody to determine the dsRNA distribution in C. sinensis. The results showed that high expression of dsRNA was distributed in the oral sucker, pharynx, gut, and body surface of C. sinensis (Fig 5E), confirming that C. sinensis could activate TLR3 via excreting dsRNA.
In addition, IL-6 ( Fig 5F) and TNF ( Fig 5G) expression increased significantly in CsEVstimulated WT BECs. IL-6 ( Fig 5F) and TNF ( Fig 5G) expression was significantly increased in TLR3 deficient BECs co-stimulated with CsEVs compared to WT BECs. Moreover, IL-4 and IFN-γ were not detected in the incubated supernatants. Next, we explored the mechanism through which TLR3 regulates the release of proinflammatory factors induced by CsEVs. We found that the phosphorylation of p65 (Fig 5H and 5I), ERK (Fig 5H and 5J), and p38 (Fig 5H  and 5K) was significantly higher in WT BECs than in cells without irritants, and further increased in TLR3 -/-BECs induced by CsEVs (Fig 5H-5K). Poly (I:C) pretreatment partially inhibited the phosphorylation of ERK (Fig 5H and 5J) and p38 (Fig 5H and 5K). P38 and ERK inhibitors were used to explore the regulatory mechanism of IL-6 and TNF expression, and the results showed that the inhibitors could significantly inhibit IL-6 ( Fig 5L) and TNF (Fig 5M) expression induced by CsEVs, respectively.

TLR3 deficiency promoted the liver lesions and myofibroblasts activation induced by CsEVs
To investigate whether TLR3 can be activated by CSEVs and participate in the pathogenesis of C. sinensis, we injected WT and TLR3 -/mice with CsEVs. The CsEVs injection significantly increased the expression of TLR3 in the liver ( Fig 6A) and promoted IL-6 ( Fig 6B), TNF ( Fig  6C) and IFN-γ expression (Fig 6D). In addition, CsEVs altered the bile duct morphology and induced inflammatory cells to accumulate, myofibroblasts activation and collagen expression in liver of WT mice (Fig 6E-6K). CsEVs result in higher IL-6 and TNF expression and lower IFN-γ expression in TLR3 -/mice (Fig 6B-6D), accompanied by more serious liver injures ( Fig  6E-6G), myofibroblast activation and collagen deposition (Fig 6H-6K).

Poly (I:C) intervention increased body weight and decreased the mortality and parasite burden due to C. sinensis infection in mice
Compared with C. sinensis-infected WT mice, poly (I:C) intervention significantly alleviated the weight loss in mice caused by C. sinensis-infection (Fig 7A). The mortality of poly (I:C) intervened C. sinensis-infection WT mice dropped by 0 from 40% (12/30) in the absence of poly (I:C) intervention in WT mice (Fig 7B). In addition, poly (I:C) intervention significantly reduced the number of intrahepatic parasites from an average of 29 parasites/mouse in WT- infected mice to an average of 8 parasites/mouse (Fig 7C), and the number of C. sinensis adults was significantly reduced (Fig 7C).

Poly (I:C) intervention reduced liver inflammation and proinflammatory cytokine release induced by C. sinensis
To investigate the effect of poly (I:C) on liver damage, pathological changes in the liver were observed, and proinflammatory cytokine production was measured. Compared with unintervened WT mice, poly (I:C)-intervened mice showed mini liver lesions (Fig 8A) with significantly reduced liver inflammation, BECs proliferation, and injury at 7, 15, and 35 dpi (Fig 8B-8D). The expression of pro-fibrotic cytokines, IL-6 ( Fig 8E), TNF ( Fig 8F) and IL-4 ( Fig 8G) was significantly reduced, but IFN-γ (Fig 8H) was significantly increased in poly (I:C)-intervened mice compared to unintervened WT mice at 7, 15, and 35 dpi. These findings indicate that poly (I:C) intervention significantly alleviated liver inflammation and lesions caused by C. sinensis.

Poly (I:C) intervention alleviated liver fibrosis caused by C. sinensis
To investigate the role of poly (I:C) in liver fibrosis caused by C. sinensis in mice, we measured the activation of fibroblasts by α-SMA immunohistochemistry and collagen deposition with Masson staining at 7, 15, and 35 dpi.
The results showed that poly (I:C) intervention significantly reduced the number of myofibroblasts in C. sinensis-infected WT mice compared to poly (I:C)-unintervened mice (Fig 9A). Statistical analysis of the positive area of myofibroblasts showed that the number of myofibroblasts in the liver of poly (I:C)-intervened mice decreased by 7.76% and 25.73% at 15 and 35 dpi, respectively (Fig 9B).
Masson staining results showed that C. sinensis-induced collagen deposition in poly (I:C)intervened WT mice was significantly reduced compared to that in poly (I:C)-unintervened mice ( Fig 9C). Poly (I:C)-intervened mice showed almost no collagen deposition around the bile duct at 7 and 15 dpi (Fig 9C and 9D). Collagen fibrotic deposition in poly (I:C)-intervened mice was significantly reduced compared with that in poly (I:C)-unintervened WT mice at 35 dpi (Fig 9C and 9D). These results suggest that poly (I:C) intervention reduced the number of positive myofibroblasts and collagen deposition caused by C. sinensis in mice.

Discussion
TLR activation provides the first line of defense in the anti-pathogen immune response. The role of TLR3 in liver diseases promptes us to investigate whether it is involved in the liver fibrosis process caused by C. sinensis [21][22][23]. To elucidate the potential mechanism of TLR3 in C. sinensis-induced liver fibrosis, liver fibrosis modeling assays were performed using TLR3-deficient or normal C57/BL6 mice in our research. The experimental data from both types of mice helped us to explore the role of TLR3 in liver fibrosis caused C. sinensis and the mechanism of host-parasite interaction. It is noteworthy that TLR3 deficiency caused severe clonorchiasis with lower survival quality, higher liver damage, and more severe liver fibrosis. using immunofluorescence. The J2 antibody was used to perform dsRNA. Scale bars = 1mm. (F, G) Secretion levels of IL-6 and TNF in the supernatants of BECs. (H) The phosphorylation of p65, ERK, and p38 in BECs challenged with CsEVs (50 μg/mL) and pretreated with poly (I:C) (30 μg/mL) was analyzed by western blotting. (I-K) Gray analysis in Fig C. (L, M) IL-6 and TNF production in the supernatant of BECs, which were pretreated with or without p38 and ERK inhibitors for 1 h, and then coincubated with CsEVs for 18 h, were measured by ELISA. Ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, multiple T-tests were performed to compare the samples.
https://doi.org/10.1371/journal.pntd.0011325.g005 This study suggests that TLR3-based treatments have a great potential for applications in C. sinensis and other parasitic liver fibrosis.
EVs produced by C. sinensis, carrying parasite information, activating the innate immune response of mouse macrophages and BECs, produce proinflammatory cytokines [9,15]. The bile ducts are the normal parasitic site of C. sinensis, and the immune response of BECs TLRs play the important role during C. sinensis infection [9,24]. We used CsEVs and BECs to simulate the interaction process between C. sinensis and host in vitro, and found that C. sinensis releases large amounts of dsRNA via CsEVs, which were recognized by host TLR3 and activated the innate immune response.
The deregulation of response of TLRs to PAMPs in BECs can lead to various liver diseases [25]. The IL-6 and TNF secreted by BECs and macrophages are the vital cytokines in C. sinensis induced liver injure and fibrosis [15,24]. The present data indicated that BECs TLR3 deficiency resulted in significantly increased IL-6 and TNF secretion induced by CsEVs, which may be an important reason for more severe liver damage and liver fibrosis in TLR3 -/mice infected with C. sinensis. And this inference was confirmed in the CsEVs injection mice. TLR3 -/mice injection with CsEVs resulted in higher IL-6 and TNF expression, leading to more severe biliary injures.
On the other hand, the infection intensity of helminths is closely related to the severity of liver lesions caused by them [26]. We found that an important result of TLR3 deficiency the α-SMA positive myofibroblasts proportion were semi-quantified in liver of mice. (J) The collagen deposition in the liver of CsEVs injected mice were observed by Masson staining. Collagen deposition was visualized by blue stripes and indicated by the black arrow. Scale bars = 50 μm. (K) Statistical analysis of the collagen positive distribution proportion was semi-quantified in liver of mice. The data were obtained from 30 mice in each group, and three independent repeated trials were conducted. Ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, multiple T-tests were performed to compare the samples.
https://doi.org/10.1371/journal.pntd.0011325.g006  IFN-γ expression. The data were obtained from 30 mice in each group, and three independent repeated trials were conducted. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA were performed to compare the samples. was a significantly higher number of infected worms, which may be another important reason for more severe liver damage and liver fibrosis in TLR3 -/mice infected with C. sinensis. PLOS NEGLECTED TROPICAL DISEASES TLR3 alleviates C. sinensis-induced liver fibrosis C. sinensis infection significantly activates the TGF-β/Smad pathway, leading to liver fibrosis [7,27]. The recombinant worm proteins rCsMF6p/HDM promote immune response and cell differentiation through the MAPK pathway [28]. However, the interaction between TGFβ/Smad and p38 in liver fibrosis induced by C. sinensis has not been clarified. Isorhamnetin protects against liver fibrosis via inhibition of TGF-β1-mediate Smad3 and p38 MAPK signaling pathways [29]. Drug-containing serum of rhubarb-astragalus reduce the protein expression of TGF-β1 and p38 MAPK and mRNA expression of SMA-α, Smad2 and Smad3 in HK-2 cells caused by the increase of TGF-β1, and the same results are found in the treatment of p38 inhibitors [30]. These studies indicate that TGF-β/Smad pathway and p38 pathway have the mutual regulatory role in the regulation of the epithelial-mesenchymal transformation and liver fibrosis. Our study clarified the mechanism by which TLR3-p38/ERK regulated cytokine expression promoted inflammation and damage, and also found that TLR3 deletion leaded to increased activation of TGF-β/Smad pathway in C. sinensis-induced liver fibrosis. We infer that TLR3-p38/ERK regulated cytokine expression may be one of the factors inducing TGF-β/ Smad pathway activation which needs to be further explored in future research. Based on this concept, we achieved promising results with TLR3 agonists for the treatment of C. sinensis. Poly (I:C), a viral dsRNA mimetic, is the most commonly used TLR3 [31]. In parasite control, poly (I:C) is used as a vaccine adjuvant to induce a multifunctional CD4 + T cell response and enhance antibody production against Plasmodium falciparum [32,33]. Total Leishmania antigens-poly (I:C) immunization resultes in good protection in mice, which is associated with decreased footpad swelling, histopathological alterations in the footpads, and parasite burdens [31]. There is no evidence that poly (I:C) can be used to control or treat liver flukes. In this study, we used poly (I:C) to treat liver fibrosis caused by C. sinensis. Poly (I:C) intervention significantly blocked the acute phase of death in mice, reduced the number of intrahepatic parasites, and alleviated C. sinensis-induced liver fibrosis, which demonstrated that poly (I:C) has great potential for application in the treatment of clonorchiasis caused by C. sinensis. Opisthorchis viverrini infection induces liver fibrosis and even cholangiocarcinoma, causing the serious disease burden in Southeast Asian countries [34]. Whether the positive contribution of poly (I:C) is also applicable to the pathogenic process of O. viverrini deserves further investigation in future studies.
In summary, a new role for TLR3 in controlling C. sinensis-induced liver fibrosis was identified (Fig 10). TLR3 deficiency resulted in severe clonorchiasis in C. sinensis-infected mice compared with WT mice. Poly (I:C) is a promising drug for clonorchiasis treatment caused by C. sinensis. Our results will help in understanding the molecular mechanisms governing the host's immune responses to C. sinensis infections and provide new information about clonorchiasis treatment.